Light-microscopy imaging methods have been developed, with which, based on a sequential locating of individual markers, particularly fluorescent molecules, sample structures can be represented, which are smaller than the diffractive resolution limit of conventional light microscopes. Such methods are for example described in WO 2006/127692 A2; DE 10 2006 021 317 B3; WO 2007/128434 A1, US 2009/0134342 A1; DE 10 2008 024 568 A1; WO 2008/091296 A2; “sub-diffraction-limit imaging by Stochastic optical reconstruction microscopy (STORM),” Nature Methods 3, 793-796 (2006), M J Rust, M. Bates, X. Zhuang; “Resolution of Lambda/10 in fluorescence microscopy using fast single molecule photo-switching”, Geisler C. et al, Appl. Phys. A, 88, 223-226 (2007). This new branch of microscopy is also referred to as locating microscopy. The methods used are known in the literature e.g. under the names (F) PALM ((Fluorescence) Photoactivation Localization Microscopy), PALMIRA (PALM with Independently Running Acquisition), GSD (IM) (Ground State Depletion Individual Molecule Return) Microscopy) or (F) STORM ((Fluorescence) Stochastic Optical Reconstruction Microscopy).
The new methods have in common that the sample structures to be imaged are prepared with point objects, so-called markers, which have two distinctive states, namely a “bright” state and a “dark” state. For example, when fluorescent dyes are used as markers, the bright state is a fluorescence-capable state and the dark state is a not fluorescence-capable state.
In preferred embodiments, as e.g. in WO 2008/091296 A2 and WO 2006/127692 A2, photoswitchable or photoactivatable fluorescent molecules are used. Alternatively, as e.g. in DE 10 2006 021 317 B3, inherent dark states of standard fluorescent molecules can be used.
For imaging sample structures with a resolution that is higher than the conventional resolution limit of the imaging optical unit, a small partial amount of the markers is now repeatedly transferred to the bright state. Thereby, the density of the markers forming this active partial amount is to be selected so that the average distance of adjacent markers in the bright and hence in the light-microscopy imaging state is greater than the resolution limit of the imaging optical unit. The markers forming the active partial amount are imaged onto a spatially resolving light detector, e.g. a CCD camera, so that a light distribution in the form of a light spot is detected, whose size is determined by the resolution limit of the optical unit.
In this manner, a large number of raw data individual images is recorded, in which respectively another active partial amount is imaged. The focus positions of the light distributions are then determined in an image evaluation process, which represent the punctiform markers in the bright state. The focus positions of the light distributions determined from the raw data individual images are then compiled in an overall presentation in the form of an overall image data set. The high-resolution overall image resulting from this overall presentation reflects the distribution of the markers.
For a representative reproduction of the sample structure to be imaged, a sufficient number of marker signals have to be detected. However, since the number of markers in the respectively active partial amount is limited by the minimum average distance, which two markers must have in the bright state, a lot of raw data individual images have to be recorded in order to image the sample structure completely. Typically, the number of raw data individual images is in a range from 10,000 to 100,000.
In addition to the above-described lateral position determination of the markers in the object plane (hereinafter also referred to as x-y-plane), a position determination in the axial direction (hereinafter also referred to as z direction) can also take place. The axial direction is thereby meant to be the direction in the optical axis of the imaging optical unit, thus the main propagation direction of light.
Three-dimensional locations are known from so-called “Particle-Tracking” experiments, as described in Kajo et al, 1994, Biophysical Journal, 67, Holtzer et al, 2007 Applied Physics Letters, 90, and Toprak et al, 2007, Nano Letters, 7 (7). They have also already been used in imaging methods based on the above-described switching and locating of individual molecules. For this purpose, Huang et al, 2008, Science, 319 and Juette et al, 2008, Nature Methods, are referred to. For the state of the art, Pavani et al., 2009, PNAS, 106, is further referred to.
A locating of a punctiform object in the z direction can in principle take place in that the change of a light spot detected on the detection surface of the camera is evaluated, which is visible when the point object moves from the optically conjugated sharpness or focal plane to the detection surface. Thereby, a point object is to be understood in the following an object whose dimensions are smaller than the diffractive resolution limit of the imaging optical unit, in particular of the detection objective. In this case, the detection objective images such an object in the form of a three-dimensional focus light distribution into the image space. The focus light distribution generates a light spot on the detection surface of the camera, which light spot is characterized by the so-called “point spread function”, that is, point-imaging function or PSF in short. If the point object is now moved in the z direction by the focus, that is, perpendicular to the focus plane, the size and the form of the PSF change. If the detection signal corresponding to the detected light spot with respect to the size and the form of the PSF is analyzed, conclusions with regard to the actual z position of the object can thus be obtained.
If the point object is located too far from the focal plane, the light spot generated on the detection surface of the camera is so blurred that the corresponding measurement signal within the conventional measurement noise is no longer perceptible. Thus, there is a region in the object space in the z direction around the central focal or focal plane, within which a point object on the detection surface generates a light spot, which is still sharp enough to be able to be evaluated for the locating of the point object in the z direction. This region containing the focal plane in the z direction is hereinafter referred to as “depth of field”.
With a three-dimensional locating, however, the fundamental problem exists that the PSF derived from a point object is symmetrical with respect to the detection surface. This means that the PSF indeed changes when the point object is moved out from the focal plane, so that the distance of the point object to the focal plane can be determined. However, the change of the PSF is symmetrical on both sides of the focal plane, so that it cannot be decided on which side of the focal plane the point object is present within the depth of field.
There are known various methods how to deal with the above-described problem. Examples are methods which are referred to in professional circles as “astigmatism” (the above-mentioned documents Kajo et al., Holtzer et al. and Huang et al.), “Bi-plane method” (see Toprak et al. and Juette et al.) and “Double helix method” (see Pavani et al.). These methods have in common that, for locating the point object in the z direction, the light spot generated on a detector for determining a parameter is analyzed and that a z position of the point object is assigned to this parameter. This association takes place by means of an association information determined in advance, which relates the parameter to the z position of the point object. For example, a magnitude characterizing the form of the light spot is considered as the parameter, as in the astigmatism method, or, as in the case of the bi-plane method, a magnitude which relates the extents of two light spots to each other, which originate from one and the same light spot and detection surfaces are generated, whose associated focal planes are offset to each other in the object space in the z direction.
A problem is now that the association information enabling an association between the parameter determined in the measurement and an axial z position determined in advance of the actual measurement is often so inaccurate that a precise determination of the z-position is not possible. The association information is thus dependent on changes in the optical properties of the sample.
Even small changes in the optical properties result in imaging errors with the high performance optics required in the localization microscopy, e.g. spherical aberrations. This has the consequence that the form of the PSF given by the light spot changes and thus the determined association information determined for example in the form of a calibration curve is no longer correct for the new optical conditions. In this case, the wrong z position is assigned to the light spot detected on the detector.
For the user, it is often difficult to introduce calibration elements as e.g. fluorescent beads into a biological sample, which shall be measured ultimately, by means of which beads the above-mentioned calibration curve can be prepared. This is especially valid when these calibration elements shall fluoresce in different colors, in order to avoid errors by the chromatic aberration.
Therefore, a preferred variant in practice is to carry out the calibration, that is, the determination of the association information, with an own calibration sample. Here, however, the problem of erroneous calibration has a particularly strong effect, as the optical properties of the calibration sample are never identical to the actual measurement sample. Small differences in the thickness of the cover glass or differences in the embedding medium of the sample can already lead to a significant deviation of the form of the calibration curve.
Even when calibration samples were introduced directly into the sample to be measured with a great experimental effort, the calibration curve determined in this manner can be faulty. For example, even small temperature changes lead to the fact that typical immersion oils change their refractive index, which in turn leads to spherical aberrations in the image.
Thus, a change in the calibration curve between the date of commencement of the calibration and the time of actual measurement may also occur in one and the same sample. In addition, the signal of a fluorescent bead used as a calibration sample certain size differs always from the signal of the point object forming single molecule, which in turn leads to an erroneous calibration.
In practice, these problems lead to the fact that accurate absolute determinations of the z position of a point object are often not possible. It is thus although quite possible to determine relative differences in the z position and thus also to separate and adjacent structures with high resolution from one another. However, a statement of how far any adjacent structures are removed from one other exactly, is difficult. It is thereby important to distinguish between the resolution, that is, the possibility to separate closely spaced structures from each other, and the absolute position determination. The association information used in the state of the art thus regularly allows for example the desired resolution in the form of a calibration curve, but not a precise determination of the absolute z position of the point object. This fact can also be described as a substantial (typically nonlinear) distortion of the three-dimensional image in the z direction, which results from the optical aberrations. Especially in modern biology, this is a big problem. For example, the exact form and arrangement of proteins influence their function dramatically. In order to obtain information regarding the structural arrangement, one is thus dependent on accurate and absolute measurements in all three spatial directions. The insufficient calibration options that exist in the state of the art for the locating in the z direction do not allow sufficient reliability.